Team:TU Darmstadt/Protocols/Dephosphorylation

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Revision as of 22:49, 16 September 2012

Dephosphorylation

About

Antarctic Phosphatase catalyzes the removal of 5' phosphate groups of DNA and RNA and thus prevents re-ligation of cut vectors. It is used before ligation.

Procedure

  1. The reaction mix contains:
    • up to 5 µg of digested DNA (no purification needed)
    • 1 µL of Antarctic Phosphatase
    • 1/10 of reaction volume of 10x Antarctic Phosphatase Reaction Buffer
    • 1 µL of Antarctic Phosphatase
  2. Incubate at 37° C for 30 minutes (3' ends) or 60 minutes (5' ends)
  3. Heat inactivate at 65° C for 15 minutes
  4. Continue with ligation
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